Trigeminally-mediated pain disorders are incompletely understood at the molecular level. TRP(V) ion channels have been implicated to function in trigeminal sensory transduction. TRPV4 is multimodally activated, e.g. by mechanical, thermal and tonicity stimuli, and products of the trpv4 gene have been detected in trigeminal ganglion sensory neurons and their peripheral projections, f/pyf'' mice, previously generated by the PI, have an elevated threshold for noxious mechanical and tonicity stimuli. The critical question remains unresolved whether and howTRPV4* trigeminal sensory neurons transduce such stimuli that elicit nociceptive behavior. Thus, the objective of this proposal is to investigate molecular and cellular mechanisms of frpv4-mediated transduction of noxious osmotic and mechanical stimuli in trigeminal sensory neurons by development of a novel mouse model. The hypothesis to be tested is whether trpv4 gene expression in trigeminal sensory neurons is critical for neurosensory transduction in response to noxious stimuli. Specifically, (1) primary cultures of trigeminal neurons from trpv4~*~ mice will be investigated by patch-clamp and Ca*+ imaging in response to tonicity and mechanical stimuli, modulated by activation of proteinase-activated receptor 2 (PAR-2), a physiologically relevant pro-algesic pathway, and for their "tonicity-tuning" of voltage-gated sodium channels (lNa) and capsaicin-response; (2) same as in Aim (1),but for dissociated sensory neurons from sensory-neuron-specific, inducible trpv4'~ mice; (3) sensdry-neuron- specific, inducible trpv4^~ mice will be generated to test the response of these mice to noxious osmotic stimuli and osmotic modulation of capsaicin stimuli of trigeminal peripherals, modulated by activation of PAR-2. For Aims (1)-(2), primary cultured neurons will be labeled by a genetically-encoded fluorescent reporter gene, expressed in a bacterialTartificial chromosometransgenic line of mice. For Aim (3), mice will be subjected to nocifensive behavioral assays and c-FOS studies of the brain stem trigeminal spinal nucleus in response to stimulation. Thus, the dependence of sensory transduction and transmission of noxious trigeminal stimuli on trpv4 will be deconstructed at the cellular level, clarifying the relative contribution of the trigeminal sensory neuron. Moreover, light will be shed on how trpv4 functions in "tonicity-tuning" of the capsaicin response and of iNa-. and on modulation of TRPV4 by PAR-2. Thus, our new mouse model will help us better understand how trigeminal ganglion neurons respond to noxious stimuli and which role the trpv4 gene plays in this process, hopefully opening up new avenues for treatment of trigeminally-mediated pain.